ABSTRACT Staphylococcus aureus is a major cause of community- and hospital-acquired diseases. Infections with antibiotic-resistant MRSA strains are associated with increased mortality. To identify targets for anti-MRSA therapy, this proposal analyzes the trafficking of surface proteins, which is essential for S. aureus colonization and invasive disease. Surface proteins are anchored to the cell wall, a process that requires specific signals and targeting mechanisms. Sortase A recognizes the LPXTG motif in C-terminal sorting signals, which are cut and covalently linked to peptidoglycan. Two types of surface protein precursors are distinguished. Precursors with canonical signal peptides are deposited in the envelope at the cell poles. Precursors with YSIRKxxxGxxS motif signal peptides traffic to the cross-wall. Confined by septal membranes during cell division, the cross-wall separates newly divided cells. It represents the site of de novo peptidoglycan synthesis and is split for completion of the cell cycle. Here we report that lipoteichoic acid (LTA) is synthesized at septal membranes and required for septal secretion of YSIRKxxxGxxS precursors. During secretion, the YSIRKx peptide is removed from the precursor. We screened temperature-sensitive mutants for defects in YSIRKxxxGxxS precursor secretion and identified variants with mutations in secA, secG and pepV. secA mutants are defective for the transport of all precursors, polar and septal secretion. secG and pepV mutants exhibit defects in septal secretion. SecA co-purifies with YSIRKxxxGxxS precursors and with PepV. We will test the hypothesis that lipid products of LTA synthesis are critical for the activation of PepV at septal membranes. Although YSIRKxxxGxxS precursors associate with SecA, we predict that precursor complexes cannot activate SecYEG translocons unless the YSIRKx peptide is removed. Upon completion of the cross-wall, LtaS, the catalyst of LTA synthesis, is cut and released while the products of LTA synthesis are dispersed until the next cell division cycle. The proposal will analyze LtaS variants defective in LTA synthesis and LtaS cleavage and characterize the contributions of specific lipids and LtaS regulation for septal secretion. We will characterize YSIRKxxxGxxS precursors bound to SecA for their ability to activate translocons in the presence of specifc lipids and PepV and study precursor mutants for their ability to activate SecYEG translocons. Last, we will characterize the subcellular distribution of PepV, its association with septal lipids, ability to cleave YSIRKxxGxxS precursors and contribution to S. aureus colonization and pathogenesis.